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Gata6 Potently Initiates Reprograming of Pluripotent and Differentiated Cells to Extraembryonic Endoderm Stem Cells

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Gata6 Potently Initiates Reprograming of Pluripotent and Differentiated Cells to Extraembryonic Endoderm Stem Cells Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells Sissy E. Wamaitha,1 Ignacio del Valle,1 Lily T.Y. Cho,1,4 Yingying Wei,2,5 Norah M.E. Fogarty,1 Paul Blakeley,1 Richard I. Sherwood,3 Hongkai Ji,2 and Kathy K. Niakan1 1Mill Hill Laboratory, The Francis Crick Institute, London NW7 1AA, United Kingdom; 2Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA; 3Brigham and Women’s Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extra- embryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subse- quent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, sug- gesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types. [Keywords: reprograming; pluripotency; mouse embryonic stem cells; human embryonic stem cells; extraembryonic endoderm; Gata6] Supplemental material is available for this article. Received December 9, 2014; revised version accepted May 21, 2015. One of the earliest specification events during mammali- et al. 2006; van den Berg et al. 2008; Zhang et al. 2008; an development is the divergence of pluripotent epiblast Hall et al. 2009; Niwa et al. 2009; Festuccia et al. 2012). progenitor (EPI) cells, which give rise to the embryo prop- In contrast, PrE and self-renewing XEN cells lack ex- er, and primitive endoderm (PrE) cells, which mainly con- pression of pluripotency genes and require an extraembry- tribute to the yolk sac. In mice, embryonic stem (ES) cells onic endoderm (ExEn) program, including transcription and extraembryonic endoderm stem (XEN) cells can be de- factors Gata4, Gata6, Sox7, and Sox17 (Soudais et al. rived from the EPI and PrE lineages, respectively, and re- 1995; Arman et al. 1998; Morrisey et al. 1998; Koutsoura- tain characteristics of their cell type of origin (Evans and kis et al. 1999; Futaki et al. 2004; Capo-Chichi et al. 2005; Kaufman 1981; Martin 1981; Kunath et al. 2005). Mouse Kunath et al. 2005; Chazaud et al. 2006; Shimoda et al. ES (mES) cells are able to self-renew and remain pluripo- 2007; Brown et al. 2010b; Morris et al. 2010; Niakan tent and depend on a gene regulatory network surround- et al. 2010; Artus et al. 2011; Schrode et al. 2014). It re- ing the core transcription factors Oct4, Sox2, and Nanog mains unclear how cells within the early embryo diverge (Boyer et al. 2005; Loh et al. 2006; Chen et al. 2008). Tran- to favor either a pluripotency or an ExEn gene regulatory scription factors such as Esrrb and Klf4 are also regulated network. The overexpression of the zinc finger transcrip- by and reinforce the core pluripotency factors (Ivanova tion factor Gata6 or Gata4 is sufficient to reprogram mES cells into XEN-like cells that contribute to PrE-de- rived lineages in vivo (Fujikura et al. 2002; Shimosato et al. 2007). The GATA factors are therefore part of a sub- Present addresses: 4Neusentis, Pfizer Worldwide Research and Develop- set of transcription factors that are capable of inducing cel- 5 ment, Great Abington, Cambridge CB21 6GS, UK; Department of Statis- lular reprograming, although precisely how this occurs tics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. Corresponding author: [email protected] Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.257071. © 2015 Wamaitha et al. This article, published in Genes & Develop- 114. Freely available online through the Genes & Development Open Ac- ment, is available under a Creative Commons License (Attribution 4.0 In- cess option. ternational), as described at http://creativecommons.org/licenses/by/4.0/. GENES & DEVELOPMENT 29:1239–1255 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/15; www.genesdev.org 1239 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Wamaitha et al. has yet to be elucidated. Ectopic expression of the tran- six transcription factors (Gata4, Gata6, Hnf4a, Foxa3, scription factor MyoD converts fibroblasts to myogenic Sox7, and Sox17) that are expressed in the PrE or its deriv- cells (Davis et al. 1987), a combination of transcription atives and are functionally required to establish or main- factors reprograms fibroblasts to induced pluripotent tain this lineage (Chen et al. 1994; Soudais et al. 1995; stem (iPS) cells (Takahashi and Yamanaka 2006), and in Molkentin et al. 1997; Kaestner et al. 1998; Morrisey vivo pancreatic exocrine cells can be reprogramed into in- et al. 1998; Koutsourakis et al. 1999; Capo-Chichi et al. sulin-secreting β-like cells (Zhou et al. 2008). Similarly, 2005; Artus et al. 2011; Schrode et al. 2014). To investigate overexpression of Cdx2 is sufficient to reprogram mES whether their expression is sufficient to induce repro- cells to trophectoderm-like cells that contribute solely graming of mES cells to iXEN cells, we used a site-specific to placental lineages in vivo (Niwa et al. 2005). However, recombination-based integration strategy (Hochedlinger the mechanisms by which single transcription factors re- et al. 2005; Beard et al. 2006) to generate mES cells ex- program existing gene expression patterns toward that of pressing a single copy of a tetracycline/doxycycline-in- their target cell type remain unclear. ducible Gata4, Gata6, Sox7, Sox17, Hnf4a,orFoxa3 Induction of the SRY homeobox gene Sox17 has also transgene. To test the fidelity of the system, we also engi- been shown to induce ExEn gene expression in mES cells neered control mES cells that induce the expression of a (Niakan et al. 2010; McDonald et al. 2014). Intriguingly, gene encoding a red fluorescent protein, dsRed. We con- SOX17 induction in human ES (hES) cells instead drives firmed robust induction above or close to levels present an embryonic endoderm program (Seguin et al. 2008). in embryo-derived XEN (eXEN) cells by quantitative This incongruence is consistent with our previous obser- RT–PCR (qRT–PCR) analysis of the Flag- or V5-tagged vations that the initial ES cell state influences differentia- transgene (Supplemental Fig. S1A). We also observed ro- tion outcomes (Cho et al. 2012). Furthermore, while bust red fluorescence in dsRed-overexpressing mES cells the induction of SOX7 drives ExEn gene expression in (Supplemental Fig. S1B). hES cells (Seguin et al. 2008), stable self-renewing human To determine the effect of these factors under condi- XEN cells have yet to be established. The effect of GATA tions that would otherwise maintain pluripotency, we in- factor induction in hES cells has not been tested, and it is duced exogenous expression with doxycycline for 6 d in unclear whether Gata6 can function as a master transcrip- the presence of LIF and serum, which are known to main- tional regulator to induce a XEN program from cells other tain mES cell self-renewal indefinitely in culture (Fig. 1A). than mES cells. Gata6 or Gata4 overexpression resulted in reprograming We developed a highly efficient approach to understand to cells with the dispersed, refractile, and stellate mor- the molecular mechanisms of Gata6-mediated reprogram- phology characteristic of eXEN (Fig. 1B) and growth fac- ing and show that Gata6 is a potent inducer of lineage re- tor-converted XEN (cXEN) cells (Kunath et al. 2005; Cho programing in multiple cell types. We demonstrate that a et al. 2012). qRT–PCR analysis of the 3′ untranslated re- short pulse of Gata6 induction is ample to perturb gene ex- gion (UTR) confirmed that Gata6-orGata4-induced cells pression in mES cells and initiate conversion to induced expressed endogenous Gata6 and Gata4 as well as key XEN (iXEN) cells, while longer induction fully down-regu- ExEn genes, including Sox7, Sox17, Lama1, Col4a1, lates the pluripotency program. Using genome-wide tran- Sparc, Dab2, Foxa3, and Hnf4a, to levels above or close scriptional and chromatin immunoprecipitation (ChIP) to those present in eXEN cells (Fig. 1C; Supplemental analyses, we found that Gata6 is able to rapidly and Fig. S1C). Immunofluorescence analysis also confirmed directly inhibit core and peripheral genes within the pluri- that both Gata4-induced (data not shown) and Gata6-in- potency regulatory network as well as directly activate an duced cells express Gata4, Gata6, Sox17, and Laminin pro- ExEn program. Despite lingering expression of Oct4 fol- teins (Fig. 1D). Moreover, Gata6-induced cells no longer lowing Gata6 induction, loss-of-function analysis sug- expressed Oct4 protein (Fig. 1D), suggesting that they gests that Oct4 is not required to drive this lineage have been reprogramed to iXEN cells. switch in mES cells. Gata6 expression in more committed In contrast, expression of Sox7, Sox17, Hnf4a,orFoxa3 neural cells also drives reprograming to iXEN-like cells. failed to induce a morphological switch to XEN-like cells We show that GATA6 induction in hES cells initiates within 6 d of induction (Fig.
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